Compounds for the treatment of cancers associated with human papillomavirus

ABSTRACT

The present invention relates to the pyrrolidine substituted with flavone derivatives, represented by the compounds of Formula (I) 
     
       
         
         
             
             
         
       
     
     or a pharmaceutically acceptable salt, a solvate, a stereoisomer or a diastereoisomer thereof for use in the treatment of cancers associated with human papillomavirus. The present invention also relates to the pharmaceutical compositions containing the compounds of Formula (I) for the treatment of cancers associated with human papillomavirus.

FIELD OF THE INVENTION

The present invention relates to the pyrrolidine substituted withflavone derivatives, represented by the compounds of Formula (I) (asdescribed herein) or pharmaceutically acceptable salts, solvates,stereoisomers or diastereoisomers thereof for use in the treatment ofcancers associated with human papillomavirus (HPV). The presentinvention also relates to pharmaceutical compositions containing thecompounds of Formula (I) for use in the treatment of cancers associatedwith human papillomavirus.

BACKGROUND OF THE INVENTION

Human papillomavirus (HPV) is a circular, non-enveloped dsDNA virus thatinfects squamous epithelial cells. HPV enters the body, usually througha break in the skin, and then infects the cells in the layers of theskin. HPV is transmitted by skin-to-skin contact. HPV infections can beacquired through a cut or through sexual activity with an infectedperson. This includes kissing or touching the skin of the infected areasand having intercourse. A mother with a genital HPV infection may alsotransmit the virus to the infant during labour.

HPVs are a group of more than 120 related viruses, 33% of which areknown to infect genital tract (Microbiology and Molecular BiologyReviews, 2004, 68 (2):362-372). Certain types of human papillomavirusare able to transform normal cells into abnormal ones which can go on toform cancer. Accordingly, these viruses are classified as high-risktypes and low-risk types. High-risk HPVs are associated with cancers.Genital HPV infections are very common and can lead to anogenitalcancers. Persistent infections with high-risk HPVs are the primary causeof cervical cancer. HPV infections also cause anal cancer, vulvarcancer, vaginal cancer and penile cancer (Int. J. Cancer, 2006, 118(12):3030-44). The high risk subtypes are HPV 16, 18, 31, 33, 35, 39, 45, 51,52, 56, 58, 59, 66 and 68, the most common being HPV 16, 18, 31, 33 and45. Further, several types of HPVs, particularly type 16, have beenfound to be associated with HPV-positive oropharyngeal cancer, a form ofhead and neck cancer (N. Engl. J. Med., 2007, 356(19):1944-56).

High risk HPVs produce two oncoproteins, E6 and E7, which are necessaryfor viral replication. During the HPV infection in humans, the HPV E6protein binds and promotes the degradation of tumor suppressor p53 by anubiquitin-mediated pathway diminishing the ability of the cell toundergo apoptosis. The HPV E7 protein binds and degrades theretinoblastoma protein (pRb), preventing it from inhibiting thetranscription factor E2F, resulting in loss of cell cycle control.

It has been estimated that HPV accounts for approximately 5% of allcancers worldwide (Int. J. Cancer, 2006, 118(12):3030-3044).

Cancer of the cervix uteri is the second most common cancer among womenworldwide. About 86% of the cases occur in developing countries.Cervical cancer accounts to 13% of the cancers occurring in females(World: Human Pappilomavirus and related cancers, summary report,November 2010).

Persistent Human papillomavirus (HPV) infections are now recognized asthe cause of essentially all cervical cancers. According to the AmericanCancer Society, in 2010, about 12,000 women in the United States wouldbe diagnosed with this type of cancer and more than 4,000 would die fromit. Cervical cancer is diagnosed in nearly half a million women eachyear worldwide, claiming a quarter of a million lives annually.

Vulvar and Vaginal cancers account to about 3 to 5% and 1-2%respectively of all gynecologic cancers and penile cancers accounts toabout 0.2% of all cancers in the United States. Despite theirinfrequency, vulvar, vaginal and penile cancers remain importantdiseases, because of their significant impact on sexuality. Though thereis no single etiologic factor, there is a strong association with HPVinfection. HPV is thought to be responsible for about 40% of penilecancers. Many studies have shown the presence of HPV types 16 and 18 inpenile carcinoma (Hum. Pathol., 1991, 22.908-913). HPV is alsoresponsible for about 65% of vaginal (International Journal of Cancer,2009, 124(7):1626-1636) and 50% of vulvar cancers (Vaccine, 2006,24(suppl 3): S11-S25) and HPV-16 accounted for most HPV positive casesfor both the cancers (Obstet. Gynecol., 2009, 113(4):917-24).

HPV infection is also associated with anal cancer. It is estimated thatabout 1,600 new cases of HPV-associated anal cancers are diagnosed inwomen and about 900 are diagnosed in men each year in the United States.In general, HPV is thought to be responsible for about 90% of analcancers (International Journal of Cancer, 2009, 124(7):1626-1636).Notably HPV 16 seems to be responsible for most of the anal cancer.According to a study HPV 16, was detected in 84 percent of anal cancerspecimens examined (New England Journal of Medicine, 1997,337(19):1350-8).

Cancer of head and neck typically refers to squamous cell carcinomas ofthe head and neck. Head and neck cancers account for approximately 3percent of all cancers in the United States (A Cancer Journal forClinicians, 2010, 60(5): 277-300). Head and neck cancers are identifiedby the area in which they begin. They are typically classified as:cancers of oral cavity, salivary gland, paranasal sinuses, nasal cavity,pharynx, nasopharynx, oropharynx, hypopharynx, larynx and lymph nodes inthe upper part of the neck. Cancer of the oral cavity (the fronttwo-thirds of the tongue, the gingiva, the buccal mucosa, the floor ofthe mouth under the tongue, the hard palate, and the small area behindthe wisdom teeth) and cancer of the oropharynx (the soft palate, thebase of the tongue, and the tonsils) are the most common types of cancercaused by HPV. Studies have shown that about 60% of oropharyngealcancers are caused by HPV (Cancer Epidemiology, Biomarkers andPrevention, 2005, 14(2):467-475), in particular HPV16, is a causalfactor for some head and neck squamous cell carcinoma (HNSCC).

HPV has also been associated with lung carcinomas. According to thepublished articles the incidence of HPV in lung cancer was 24.5% (LungCancer, 2009, 65: 13-18). A study conducted in China revealed that therisk of lung squamous cell carcinomas was 3.5 times higher amongHPV-positive population compared with the HPV-negative population and16.9 times higher for patients with positive HPV-16 than negative HPV-16(Oncol. Rep., 2009, 21(6):1627-32).

A role for human papillomaviruses has also been proposed in a diverserange of other malignancies, particularly, non-melanoma skin cancer thecommonest malignancy in fair-skinned populations worldwide. Skin canceris rarely fatal and is responsible for less than 1% of all cancerdeaths. However its impact on the public health is neverthelessconsiderable. The involvement of HPV in human skin cancer was firstdemonstrated in patients with the rare hereditary diseaseepidermodysplasia verruciformis (Journal of National Cancer InstituteMonographs, 2003, No. 31).

HPV is also associated with intraepithelial neoplasia of the conjunctiva(0-80%) and in 62-100% of invasive carcinomas of the conjunctiva, eyelidand lacrimal sac (IARC Monographs, 64:130). There is a strongassociation between HPV and conjunctival papillomas. HPV type 6/11 isthe most common HPV type in conjunctival papilloma (Br. J. Ophthalmol.,2001, 85:785-787).

HPV 6, 11 and 13 are typically labeled as low-risk, because theinfection with these types has low oncogenic potential and usuallyresults in the formation of benign lesions such as genital warts(technically known as condylomata acuminata) and mild dysplasia of thecervix, HPV 6 and 11 are also associated with conjunctival papilloma.

Despite the high incidence of genital HPV infection and its associationwith malignant diseases, there is no effective antiviral therapy for HPVinfection. Gardasil® and Cervarix® are the two vaccines currently on themarket against two of the most common high risk HPVs (HPV 16 and 18).However, they are only of prophylactic type and do not treat theexisting HPV associated cancer. Moreover, the high cost, issues withsocial acceptance, and limitations in health care systems through whichthe vaccine can be provided, limits the availability of this vaccine towomen, particularly in developing countries where HPV-associatedanogenital cancers (relating to the anus and the genitals) are mostcommonly found. Consequently there remains a need to identify other,less expensive and more universally available approaches for preventingand/or treating HPV associated cancers.

The inventors have surprisingly found that pyrrolidine substituted withflavone derivatives are effective against cancers associated with HPV.

The invention described herein provides pyrrolidine substituted withflavone derivatives represented by Formula (I) (as described herein) forthe treatment of human papillomavirus associated cancers.

SUMMARY OF THE INVENTION

According to one aspect of the invention, there is provided use of acompound of Formula (I), a pharmaceutically acceptable salt, a solvate,a stereoisomer or a diastereoisomer thereof for the treatment of acancer associated with human papillomavirus.

According to another aspect of the invention, there is provided use of acompound of Formula (I), a pharmaceutically acceptable salt, a solvate,a stereoisomer or a diastereoisomer thereof for the treatment of acancer associated with HPV wherein the cancer is anal cancer, vulvarcancer, vaginal cancer, penile cancer, cervical cancer, head and neckcancer such as oropharyngeal cancer and cancer of the oral cavity, lungcancer, non-melanoma skin cancer or cancer of the conjunctiva.

According to yet another aspect of the invention, there is provided useof a compound of Formula (I), a pharmaceutically acceptable salt, asolvate, a stereoisomer or a diastereoisomer thereof for the treatmentof cervical cancer.

According to another aspect of the invention, there is provided a methodfor the treatment of human papillomavirus associated cancers, comprisingadministering to the subject in need thereof a therapeutically effectiveamount of a compound of Formula (I) a pharmaceutically acceptable salt,a solvate, a stereoisomer or a diastereoisomer thereof.

According to another aspect of the invention, there is provided a methodfor the treatment of a cancer associated with HPV wherein the cancer isselected from anal cancer, vulvar cancer, vaginal cancer, penile cancer,cervical cancer, head and neck cancer such as oropharyngeal cancer andcancer of the oral cavity, lung cancer, non-melanoma skin cancer orcancer of the conjunctiva in a subject, comprising administering to thesubject in need thereof a therapeutically effective amount of a compoundof Formula (I), a pharmaceutically acceptable salt, a solvate, astereoisomer or a diastereoisomer thereof.

According to yet another aspect of the invention, there is provided amethod for the treatment of cervical cancer, comprising administering tothe subject in need thereof a therapeutically effective amount of acompound of Formula (I), a pharmaceutically acceptable salt, a solvate,a stereoisomer or a diastereoisomer thereof.

According to another aspect of the invention there is provided apharmaceutical composition, comprising a therapeutically effectiveamount of a compound of Formula (I) a pharmaceutically acceptable salt,a solvate, a stereoisomer or a diastereoisomer thereof in associationwith a pharmaceutically acceptable carrier for the treatment of a cancerassociated with HPV.

According to another aspect of the invention, there is provided a methodfor the manufacture of medicaments, comprising a compound of Formula(I), a pharmaceutically acceptable salt, a solvate, a stereoisomer or adiastereoisomer thereof which are useful for the treatment of a cancerassociated with HPV.

Other aspects and further scope of applicability of the presentinvention will become apparent from the detailed description to follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts the reproductions of Western blots showing significantupregulation of p53 in the cells treated with compound A and compound B.

FIG. 1B depicts the reproductions of Western blots showing significantdownregulation of expression of E6 and E7 in SiHa cells treated withcompound A and compound B.

FIG. 2 depicts the reproductions of RT-PCR showing significantdownregulation of expression of E6 and E7 in SiHa and HeLa cells treatedwith compound A and compound B at the transcription level.

FIG. 3A is a graphical representation of p53-EGFP nuclear translocationin SiHa cells.

FIG. 3B is a graphical representation of p53-EGFP nuclear translocationin HeLa cells.

FIG. 4 is a graphical representation of tumor growth profile in SiHaxenograft animals after administration of Compound A.

FIG. 5 is a graphical representation of tumor growth profile in SiHaxenograft animals after administration of Compound B.

DETAILED DESCRIPTION OF THE INVENTION

The general terms used hereinbefore and hereinafter preferably havewithin the context of this disclosure the following meanings, unlessotherwise indicated. Thus, the definitions of the general terms as usedin the context of the present invention are provided herein below:

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, as wellas represents a stable compound, which does not readily undergotransformation such as rearrangement, cyclization, elimination, etc.

The term “C₁-C₄alkyl” refers to the radical of saturated aliphaticgroups, including straight or branched-chain containing from 1 to 4carbon atoms. Examples of alkyl groups include but are not limited tomethyl, ethyl, propyl, butyl, isopropyl, isobutyl, sec-butyl, tert-butyland the like.

The term “C₁-C₄alkoxy” refers to an alkyl group as defined aboveattached via oxygen linkage to the rest of the molecule. Examples ofalkoxy include, but are not limited to methoxy, ethoxy, propoxy, butoxy,tert-butoxy and the like.

The term “halogen” refers to fluorine, chlorine, bromine and iodine.

The term “hydroxy” or “hydroxyl” as used herein, refers to —OH group.

The term “therapeutically effective amount”, as used herein refers tothe amount of a compound represented by Formula (I), a pharmaceuticallyacceptable salt, a solvate, a stereoisomer or a diastereoisomer thereof,that, when administered to a subject in need of such treatment, issufficient to inhibit the activity of human papillomavirus (HPV) suchthat the disease mediated by HPV is reduced, treated or alleviated.

The term “HPV” or “Human papillomavirus” as used herein refers to amember of the papillomavirus family of viruses that is capable ofinfecting mammals. The term includes both high-risk type and low risktype HPVs unless otherwise indicated.

The term “subject” as used herein, refers to an animal, preferably amammal, most preferably a human, who is in the need of treatment ofdiseases mediated by HPV. The term subject may be interchangeably usedwith the term patient in the context of the present invention.

The term “mammal” as used herein is intended to encompass humans, aswell as non-human mammals which are susceptible to infection by humanpapillomavirus. Non-human mammals include but are not limited todomestic animals, such as cows, pigs, horses, dogs, cats, rabbits, ratsand mice, and non-domestic animals.

The term “treat” or “treatment” or “treated” with reference to HPVassociated cancer in a subject, preferably a mammal, more preferably ahuman include: (i) inhibition of cancer i.e., arresting the developmentof the cancer; (ii) reduction in the regression of the cancer or slowingdown of the cancer; (iii) amelioration of the cancer i.e., reducing theseverity of the symptoms associated with the cancer (iv) relief, to someextent, of one or more symptoms associated with cancer.

As used herein the term “pharmaceutically acceptable” is meant that thecarrier, diluent, excipients, and/or salt must be compatible with theother ingredients of the formulation, and not deleterious to therecipient thereof. “Pharmaceutically acceptable” also means that thecompositions or dosage forms are within the scope of sound medicaljudgment, suitable for use for an animal or human without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

The present invention furthermore includes all solvates of the compoundsof the formula (I), for example hydrates, and the solvates formed withother solvents of crystallization, such as alcohols, ethers, ethylacetate, dioxane, dimethylformamide or a lower alkyl ketone, such asacetone, or mixtures thereof. Certain compounds of the present inventioncan exist in unsolvated forms as well as solvated forms, includinghydrated forms. Certain compounds of the present invention may exist inmultiple crystalline or amorphous forms. In general, all physical formsare equivalent for the uses contemplated by the present invention andare intended to be within the scope of the present invention.

The term “p53” refers to a nuclear phosphoprotein which acts as a tumorsuppressor.

The term “pRb” or “retinoblastoma protein” refers to a tumor suppressorprotein that is dysfunctional in many types of cancer.

The term “downregulation” refers to reducing, partially or totally, theindicated activity or expression. In the context of the presentinvention the term “downregulation of the oncoproteins E6 and E7” refersto the decrease in the expression level of E6 and E7 respectively. Thelevel may be determined by any suitable method in the art, includingWester blot assay.

The term “upregulation of p53” refers to increase in the expressionlevel of the tumor suppressor protein 53.

The term “senescence” refers to the stage in which the cells lose theirability to divide.

The term “apoptosis” refers to the natural process of programmed celldeath. It is a process of self-destruction, in which the cell usesspecialized cellular machinery to kill itself. The cells disintegrateinto membrane-bound particles that are then eliminated by phagocytosis.Apoptosis is a mechanism that enables metazoans to control cell numberand eliminate cells that threaten the animal's survival.

The term “transcription” refers to a process of creating a complementaryRNA copy of a sequence of DNA.

The term “cell cycle arrest” refers to a regulatory process that haltsthe progression through the cell cycle during one of the normal phases(G1, S, G2, M).

The compound with anti-cancer activity refers to a substance which iscapable of inhibiting cell proliferation or is capable of inducing celldeath. Non-limiting examples of anti-cancer agent suitable for use inthe compositions of the invention include (i) microtubule disruptingagents such as taxanes and paclitaxel (ii) kinase inhibitors such asimatinib, erlotinib and BAY-43-9006, (iii) mTOR inhibitors such asrapamycin (iv) antineoplastic agents such as carboplatin, cisplatin,oxaliplatin, etoposide and Dacarbazine and (xvi) topoisomeraseinhibitors such as topotecan and irinotecan (vi) antimetabolites such ascytarabine, fluorouracil, gemcitabine, topotecan, Hidroxyurea,Thioguanine, Methotrexate (vii) antibiotics such cytotoxic agentsdoxorubicin, bleomycin and dactinomycin.

According to one aspect of the present invention there is provided acompound of Formula (I),

wherein Ar is a phenyl group, which is unsubstituted or substituted by1, 2, or 3 identical or different substituents selected from: halogen,nitro, cyano, C₁-C₄-alkyl, trifluoromethyl, hydroxyl or C₁-C₄-alkoxy; ora pharmaceutically acceptable salt, a solvate, a stereoisomer or adiastereoisomer thereof, for use in the treatment of a cancer associatedwith HPV.

According to one aspect of the invention there is provided a (+)-transisomer of the compound of Formula (I), as indicated in Formula (IA)below,

wherein Ar is a phenyl group, which is unsubstituted or substituted by1, 2, or 3 identical or different substituents selected from halogen,nitro, cyano, C₁-C₄-alkyl, trifluoromethyl, hydroxyl or C₁-C₄-alkoxy; ora pharmaceutically acceptable salt thereof or a solvate thereof for usein the treatment of a cancer associated with HPV.

According to another aspect of the invention there is provided acompound of Formula (I), a pharmaceutically acceptable salt, a solvate,a stereoisomer or a diastereoisomer thereof, wherein Ar is phenyl groupsubstituted by 1, 2, or 3 identical or different substituents selectedfrom chlorine, bromine, fluorine, iodine, C₁-C₄-alkyl ortrifluoromethyl, for use in the treatment of a cancer associated withHPV.

According to another aspect of the invention there is provided acompound of Formula (I), a pharmaceutically acceptable salt, a solvate,a stereoisomer or a diastereoisomer thereof, wherein Ar is phenyl groupsubstituted by 1, 2, or 3 identical or different halogens selected fromchlorine, bromine, fluorine or iodine, for use in the treatment of acancer associated with HPV.

According to another aspect of the invention there is provided acompound of Formula (I), a pharmaceutically acceptable salt, a solvate,a stereoisomer or a diastereoisomer thereof, wherein Ar is phenyl groupsubstituted by chlorine, for use in the treatment of a cancer associatedwith HPV.

According to another aspect of the invention there is provided acompound of Formula (I), a pharmaceutically acceptable salt, a solvate,a stereoisomer or a diastereoisomer thereof, wherein Ar is phenyl groupsubstituted by chlorine and trifluromethyl, for use in the treatment ofa cancer associated with HPV.

It will be appreciated by those skilled in the art that the compounds ofFormula (I) contain at least two chiral centres and hence, exists in theform of two different optical isomers (i.e., (+) or (−) enantiomers),two different geometric isomers (cis and trans) and 4 differentdiasteroisomers. All such enantiomers, geometric isomers,diasteroisomers and mixtures thereof including racemic mixtures areincluded within the scope of the invention. The enantiomers of thecompound of Formula (I) can be obtained by methods disclosed in PCTApplication Publication Nos. WO2004004632, WO2007148158 and WO2008007169incorporated herein by reference or the enantiomers of the compound ofFormula (I) can also be obtained by methods well known in the art, suchas chiral HPLC and enzymatic resolution. Alternatively, the enantiomersof the compounds of Formula (I) can be synthesized by using opticallyactive starting materials.

The manufacture of the compounds of Formula (I), which may be in theform of pharmaceutically acceptable salts, and the manufacture ofpharmaceutical composition suitable for oral, rectal and/or parenteraladministration containing the above compounds are generally disclosed inPCT Application Publication No. WO2004004632, which is incorporatedherein by reference.

As indicated herein above the compound of Formula (I) may be used in theform of their salts. Preferred salt of compounds of Formula (I) includeacetates, alginates, ascorbates, aspartates, benzoates,benzenesulfonates, bisulfates, borates, cinnamates, citrates,ethanesulfonates, fumarates, glucuronates, glutamates, glycolates,hydrochlorides, hydrobromides, hydrofluorides, ketoglutarates, lactates,maleates, malonates, mesylate, nitrates, oxalates, palmoates,perchlorates, phosphates, picrates, salicylates, succinates, sulfamate,sulfates, tartrates, tosylate, trifluoroacetic acid salt and other acidaddition salts known to the person skilled in the art.

Accordingly, an aspect of the invention, the compound of Formula (IA)for use in the treatment of HPV associated cancer is selected from(+)-trans-2-(2-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxy-methyl-1-methyl-pyrrolidin-3-yl)-chromen-4-onehydrochloride (referred to herein as compound A) or(+)-trans-3-[2[(2-Chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-chromen-4-onehydrochloride (referred to herein as compound B). Compounds A and B aredisclosed in WO2007148158 and specifically as Example 10 and Example 44,respectively.

The compound of Formula (IA) for use in the treatment of HPV associatedcancer can be(+)-trans-2-(2-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxy-methyl-1-methyl-pyrrolidin-3-yl)-chromen-4-onehydrochloride (compound A).

The compound of Formula (IA) for use in the treatment of HPV associatedcancer can be(+)-trans-3-[2[(2-Chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-chromen-4-onehydrochloride (compound B).

The cancer associated with HPV is selected from the group consisting ofanal cancer, vulvar cancer, vaginal cancer, penile cancer, cervicalcancer, head and neck cancer such as oropharyngeal cancer and cancer ofthe oral cavity, lung cancer, non-melanoma skin cancer and cancer of theconjunctiva.

A compound of Formula (I), a pharmaceutically acceptable salt, asolvate, a stereoisomer or a diastereoisomer thereof can be used for thetreatment of cervical cancer.

A method for the treatment of human papillomavirus associated cancer,comprising administering to the subject in need thereof atherapeutically effective amount of a compound of Formula (I) apharmaceutically acceptable salt, a solvate, a stereoisomer or adiastereoisomer thereof is described.

HPV associated cancers selected from the group consisting of analcancer, vulvar cancer, vaginal cancer, penile cancer, cervical cancer,head and neck cancer such as oropharyngeal cancer and cancer of the oralcavity, lung cancer, non-melanoma skin cancer and cancer of theconjunctiva in a subject, can be treated by administering to the subjectin need thereof a therapeutically effective amount of a compound ofFormula (I), a pharmaceutically acceptable salt, a solvate, astereoisomer or a diastereoisomer thereof.

Cervical cancer can be treated by administering to the subject in needthereof a therapeutically effective amount of a compound of Formula (I),a pharmaceutically acceptable salt, a solvate, a stereoisomer or adiastereoisomer thereof.

The invention provides a method of inhibiting HPV associated cancer;comprising contacting the HPV infected cells with a compound of Formula(I).

According to an aspect of the invention there is provided a method ofinhibiting high risk HPV selected from HPV 16, 18, 31, 33, 35, 39, 45,51, 52, 56, 58, 59, 66 and 68, comprising contacting the HPV infectedcells with a compound of Formula (I).

In an embodiment there is provided a method of inhibiting HPV 16, 18 and31, comprising contacting the HPV infected cells with a compound ofFormula (I).

According to this invention, the oncoproteins on HPV can bedownregulated. The oncoproteins may be the oncoproteins E6 and E7 onHPV. The downregulation can be carried out by, administering to thesubject a therapeutically effective amount of a compound of Formula (I).Inhibiting oncoproteins E6 and E7 on HPV is a means of inducing cellcycle arrest, senescence or apoptosis.

The oncoproteins E6 and E7 on HPV can be downregulated attranscriptional level.

The phosphoprotein p53 can be activated by administering atherapeutically effective amount of a compound of Formula (I) to thesubject in need thereof.

According to yet another embodiment of the present invention there isprovided a method of treatment of a cancer associated with HPV 16comprising administering to the subject in need thereof atherapeutically effective amount of a compound of Formula (I), apharmaceutically acceptable salt, a solvate, a stereoisomer or adiastereoisomer thereof.

According to another aspect of the invention, there is provided a methodfor the manufacture of medicaments, comprising a compound of Formula(I), a pharmaceutically acceptable salt, a solvate, a stereoisomer or adiastereoisomer thereof which are useful for the treatment of a cancerassociated with HPV.

There is provided a pharmaceutical composition which comprises atherapeutically effective amount of compound of Formula (I), apharmaceutically acceptable salt, a solvate, a stereoisomer, or adiastereoisomer thereof in association with a pharmaceuticallyacceptable carrier. The composition can include at least one furtherpharmaceutically active compound, wherein the further pharmaceuticallyactive compound has anticancer activity. The pharmaceutically activecompound can be selected from, but not limited to, bleomycin, cispaltin,topotecan hydrochloride, imiquimod, podofilox, trichloroacetic acid andthe like.

The pharmaceutical preparations may contain about 1 to 99%, for example,about 5 to 70%, or from about 5 to about 30% by weight of the compoundof the Formula (I) or pharmaceutically acceptable salt thereof. Theamount of the active ingredient of the Formula (I) or pharmaceuticallyacceptable salt thereof in the pharmaceutical preparations normally isfrom about 1 to 1000 mg.

The compound of Formula (I) may be administered orally, intravaginally,vulvovaginally, rectally, topically or parenterally (includingintravenous, subcutaneous, intramuscular, intravascular or infusion).The compound of Formula (I) may have to be administered by any routeappropriate to the condition to be treated. It will be appreciated thatthe preferred route may vary with the condition of the patient.

Compositions intended for pharmaceutical use may be prepared accordingto any method known in the art for the manufacture of pharmaceuticalcompositions, e.g. Remington—The Science and Practice of Pharmacy(21^(st) Edition) (2005), Goodman & Gilman's The Pharmacological Basisof Therapeutics (11^(th) Edition) (2006) and Ansel's PharmaceuticalDosage Forms and Drug Delivery Systems (9^(th) Edition), edited by Allenet al., Lippincott Williams & Wilkins, (2011), Solid-State Chemistry ofDrugs (2^(nd) Edition) (1999), each of which is hereby incorporated byreference.”

The compositions described herein may be in a form suitable for oraladministration, for example as a tablet or capsule; for parenteralinjection (including intravenous, subcutaneous, intramuscular,intravascular or infusion) for example as a sterile solution, suspensionor emulsion; for topical administration for example as an ointment,cream, gel, lotions or collodion; for rectal, vaginal or vulvovaginaladministration for example as a suppository, tampons, pessaries, creams,gels, paste, foam or vaginal ring. The composition can be administeredtopically to external surfaces of skin surface, such as vulva and/or tosurrounding areas of skin. In addition or alternatively, the compositioncan be administered intravaginally.

For oral use, the compound of Formula (I) may be administered, forexample, in the form of tablets or capsules, powders, dispersiblegranules, or cachets, or as aqueous solutions or suspensions. In thecase of tablets for oral use, carriers which are commonly used includelactose, corn starch, magnesium carbonate, talc, and sugar, andlubricating agents such as magnesium stearate are commonly added. Fororal administration in capsule form, useful carriers include lactose,corn starch, magnesium carbonate, talc and sugar.

For intramuscular, intraperitoneal, subcutaneous and intravenous use,sterile solutions of compound of Formula (I) are usually employed, andthe pH of the solutions should be suitably adjusted and buffered.

For ointments, creams, the compound of Formula (I) is formulated inoil-in-water or water-in-oil base. A vaginal cream can be administeredto contact a mucosal surface in the vaginal cavity.

For rectal or vaginal use, the compound of Formula (I) can beadministered in the form of suppositories. A suppository comprises ofcompound of Formula (I), a suitable suppository base and additives suchas preservatives, antioxidants, emulsifiers and the like. Suitablesuppository bases include natural or synthetic triglycerides or paraffinhydrocarbons. The vaginal use the compound of Formula (I) can also beadministered in the form of vaginal cream.

Compositions for oral delivery may be in the form of tablets, lozenges,aqueous or oily suspensions, granules, powders, emulsions, capsules,syrups, or elixirs. Orally administered compositions may contain one ormore optional agents, for example, sweetening agents such as fructose,aspartame or saccharin; flavoring agents such as peppermint, oil ofwintergreen, or cherry; coloring agents; and preserving agents, toprovide a pharmaceutically palatable preparation. Selectively permeablemembranes surrounding an osmotically active driving compound are alsosuitable for oral administration of compounds of present invention. Oralcompositions can include standard vehicles such as mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate, etc. Such vehicles are preferably of pharmaceutical grade.

Further, the effect of the compounds of the present invention containedin the pharmaceutical composition may be delayed or prolonged by properformulation. For example, a slowly soluble pellet of the compound may beprepared and incorporated in a tablet or capsule. The technique may beimproved by making pellets of several different dissolution rates andfilling capsules with a mixture of the pellets. Tablets or capsules maybe coated with a film which resists dissolution for a predictable periodof time. Even the parenteral preparations may be made long-acting, bydissolving or suspending the compound in oily or emulsified vehicleswhich allow it to disperse only slowly in the serum.

Compositions for rectal administration or vaginal administration aresuppositories. Suppositories are solid bodies for insertion into therectum or vagina which melt or soften at body temperature releasing oneor more pharmacologically or therapeutically active ingredients.Pharmaceutically acceptable substances utilized in rectal suppositoriesare bases or vehicles and agents to raise the melting point. Examples ofbases include cocoa butter (theobroma oil), glycerin-gelatin, carbowax(polyoxyethylene glycol) and appropriate mixtures of mono-, di- andtriglycerides of fatty acids. Combinations of the various bases may beused. Agents to raise the melting point of suppositories includespermaceti and wax. Suppositories may be prepared either by thecompressed method or by molding.

Effective dose of the compound of Formula (I) depends at least on thenature of the condition being treated, the mode of delivery, and thepharmaceutical formulation, and will be determined by the clinicianusing conventional dose escalation studies. It can be expected to befrom about 0.1 to about 100 mg/kg body weight per day; particularly,from about 0.1 to about 10 mg/kg body weight per day; more particularly,from about 0.1 to about 5 mg/kg body weight per day.

Compounds of Formula (I) may be prepared according to the methodsdisclosed in PCT Patent Publication No. WO2004004632 and PCT PatentPublication No. WO2007148158 which are incorporated herein by reference.

The general process for the preparation of compounds of Formula (I), ora pharmaceutically acceptable salt thereof, comprises the followingsteps:

(a) treating the resolved enantiomerically pure (−)-trans enantiomer ofthe intermediate compound of Formula VIA,

with acetic anhydride in the presence of a Lewis acid catalyst to obtaina resolved acetylated compound of Formula VIIA,

(b) reacting the resolved acetylated compound of Formula VIIA with anacid of Formula ArCOOH or an acid chloride of Formula ArCOCl or an acidanhydride of Formula (ArCO)₂O or an ester of Formula ArCOOCH₃, whereinAr is as defined hereinabove in reference to the compound of Formula(I), in the presence of a base and a solvent to obtain a resolvedcompound of Formula VIIIA;

(c) treating the resolved compound of Formula VIIIA with a base in asuitable solvent to obtain the corresponding resolved β-diketonecompound of Formula IXA;

wherein Ar is as defined above;(d) treating the resolved β-diketone compound of Formula IXA with anacid such as hydrochloric acid to obtain the corresponding cyclizedcompound of Formula XA,

(e) subjecting the compound of Formula XA to dealkylation by heating itwith a dealkylating agent at a temperature ranging from 120-180° C. toobtain the (+)-trans enantiomer of the compound of Formula (I) and,optionally, converting the subject compound into its pharmaceuticallyacceptable salt.

The Lewis acid catalyst utilized in the step (a) above may be selectedfrom: BF₃, Et₂O, zinc chloride, aluminium chloride and titaniumchloride.

The base utilized in the process step (b) may be selected fromtriethylamine, pyridine and a DCC-DMAP combination (combination ofN,N′-dicyclohexyl carbodiimide and 4-dimethylaminopyridine).

It will be apparent to those skilled in the art that the rearrangementof the compound of Formula VIIIA to the corresponding β-diketonecompound of Formula IXA is known as a Baker-Venkataraman rearrangement(J. Chem. Soc., 1933, 1381 and Curr. Sci., 1933, 4, 214).

The base used in the process step (c) may be selected from: lithiumhexamethyl disilazide, sodium hexamethyldisilazide, potassiumhexamethyldisilazide, sodium hydride and potassium hydride. A preferredbase is lithium hexamethyl disilazide.

The dealkylating agent used in process step (e) for the dealkylation ofthe compound of Formula IXA may be selected from: pyridinehydrochloride, boron tribromide, boron trifluoride etherate andaluminium trichloride. A preferred dealkylating agent is pyridinehydrochloride.

Preparation of the starting compound of Formula VIA involves reacting1-methyl-4-piperidone with a solution of 1,3,5-trimethoxybenzene inglacial acetic acid, to yield1-methyl-4-(2,4,6-trimethoxyphenyl)-1,2,3,6-tetrahydropyridine, which isreacted with boron trifluoride diethyl etherate, sodium borohydride andtetrahydrofuran to yield1-methyl-4-(2,4,6-trimethoxyphenyl)piperidin-3-ol. Conversion of1-methyl-4-(2,4,6-trimethoxyphenyl)piperidin-3-ol to the compound ofFormula VIA involves converting the hydroxyl group present on thepiperidine ring of the compound, 1-methyl-4-(2,4,6-trimethoxyphenyl)piperidin-3-ol to a leaving group such as tosyl, mesyl, triflate orhalide by treatment with an appropriate reagent such asp-toluenesulfonylchloride, methanesulfonylchloride, triflic anhydride orphosphorous pentachloride in the presence of oxygen nucleophiles such astriethylamine, pyridine, potassium carbonate or sodium carbonate,followed by ring contraction in the presence of oxygen nucleophiles suchas sodium acetate or potassium acetate in an alcoholic solvent such asisopropanol, ethanol or propanol.

It is to be understood that the invention may assume various alternativevariations and step sequences, except where expressly specified to thecontrary.

Moreover, other than in any operating examples, or where otherwiseindicated, all numbers expressing, for example, quantities ofingredients used in the specification and claims are to be understood asbeing modified in all instances by the term “about”.

Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired properties to beobtained by the present invention. At the very least, and not as anattempt to limit the application of the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Those skilled in the art will recognize that several variations arepossible within the scope and spirit of this invention. The inventionwill now be described in greater detail by reference to the followingnon-limiting examples. The following examples further illustrate theinvention but, of course, should not be construed as in any way limitingits scope.

EXEMPLIFICATION

In the following examples and elsewhere, abbreviations have thefollowing meanings:

List of abbreviations ATCC American Type Cell Culture bp Base pairs BF₃boron trifluoride CO₂ carbon dioxide cDNA complementary DNA DMEMDulbecco's Modified Eagle Medium DMSO dimethyl sulfoxide DNAdeoxyribonucleic acid dsDNA double stranded Deoxyribonucleic Acid dNTPsDeoxynucleotide Triphosphates e.e enantiomeric excess EGFP enhancedgreen fluorescent protein Et₂O diethyl ether FBS fetal bovine serum gGram H hour(s) HCl hydrochloric acid HPLC high performance liquidchromatography HRP horseradish peroxidase ip intraperitoneally IPAisopropyl alcohol MeOH Methanol μg Microgram μL microlitre mg milligrammL milliliter mm millimetre mmol or mM millimolar mpk milligram perkilogram MgCl₂ magnesium Chloride MMLV-RT Moloney Murine Leukemia virusReverse Transcriptase Na₂CO₃ sodium carbonate ng nanogram NP-40 nonylphenoxypolyethoxylethanol NaF sodium fluoride Na₃VO₄ sodiumorthovanadate NaCl sodium chloride nm Nanometers pmol Picomolar PCRpolymerase chain reaction p.o per oral RNA ribonucleic Acid RT-PCRReverse transcription polymerase chain reaction SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis TFA trifluoroacetic acid UVUltraviolet

Example 1 A) Preparation of(+)-trans-2-(2-Chlorophenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-chromen-4-onehydrochloride (Compound A)

Molten pyridine hydrochloride (4.1 g, 35.6 mmol) was added to(+)-trans-2-(2-chloro-phenyl)-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-5,7-dimethoxy-chromen-4-one(0.4 g, 0.9 mmol) and heated at 180° C. for 1.5 h. The reaction mixturewas cooled to 25° C., diluted with MeOH (10 mL) and basified usingNa₂CO₃ to pH 10. The mixture was filtered and the organic layer wasconcentrated. The residue was suspended in water (5 mL), stirred for 30minutes filtered and dried to obtain the compound,(+)-trans-2-(2-chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-chromen-4-one.

Yield: 0.25 g (70%);

IR (KBr): 3422, 3135, 1664, 1623, 1559 cm⁻;

1H NMR (CDCl₃, 300 MHz): δ 7.56 (d, 1H), 7.36 (m, 3H), 6.36 (s, 1H),6.20 (s, 1H), 4.02 (m, 1H), 3.70 (m, 2H), 3.15 (m, 2H), 2.88 (m, 1H),2.58 (s, 3H), 2.35 (m, 1H), 1.88 (m, 1H); MS (ES+): m/z 402 (M+1);

Analysis: C₂₁H₂₀ClNO₅; C, 62.24 (62.71); H, 5.07 (4.97); N, 3.60 (3.48);Cl, 9.01 (8.83).

The compound (0.2 g, 0.48 mmol) as obtained above was suspended in IPA(5 mL) and 3.5% HCl (25 mL) was added. The suspension was heated to geta clear solution. The solution was cooled and filtered to obtain thecompound,(+)-trans-2-(2-Chlorophenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)chromen-4-onehydrochloride.

Yield: 0.21 g (97%); mp: 188-192° C.; [α] D25=+21.3° (c=0.2, methanol);

1H NMR (CD₃OD, 300 MHz): δ 7.80 (d, 1H), 7.60 (m, 3H), 6.53 (s, 1H),6.37 (s, 1H), 4.23 (m, 1H), 3.89 (m, 2H), 3.63 (m, 1H), 3.59 (dd, 1H),3.38 (m, 1H), 2.90 (s, 3H), 2.45 (m, 1H), 2.35 (m, 1H); MS (ES+): m/z402 (M+1)(free base).

This compound was subjected to chiral HPLC. Chiral HPLC was done usingcolumn Chiralcel OD-H (250×4.6 mm) and solvent system haxane:ethanol(92:08) with TFA (0.4%). The results are recorded at 264 nm with solventflow rate of 1 mL/minute The chiral HPLC showed 100% e.e of thecompound,(+)-trans-2-(2-chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxy-methyl-1-methyl-pyrrolidin-3-yl)-chromen-4-onehydrochloride.

B) Preparation of(+)-trans-2-(2-chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-chromen-4-onehydrochloride (Compound B)

A mixture of the compound,(+)-trans-2-(2-Chloro-4-trifluoromethylphenyl)-8-(2-hydroxymethyl-1-methylpyrrolidin-3-yl)-5,7-dimethoxy-chromen-4-one (0.25 g, 0.5 mmol),pyridine hydrochloride (0.25 g, 2.16 mmol) and a catalytic amount ofquinoline was heated at 180° C. for a period of 2.5 h. The reactionmixture was diluted with methanol (25 mL) and basified with solid Na₂CO₃to pH 10. The reaction mixture was filtered, and washed with methanol.The organic layer was concentrated and the residue purified by columnchromatography using 0.1% ammonia and 4.5% MeOH in chloroform as eluentto yield the compound,(+)-trans-2-(2-chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydroxy-methyl-1-methylpyrrolidin-3-yl)-chromen-4-one,as a yellow solid.

Yield: 0.15 g (63.7%);

¹H NMR (CDCl₃, 300 MHz): δ 7.99 (m, 2H), 7.83 (d, 1H), 6.65 (s, 1H),6.41 (s, 1H), 4.24 (m, 1H), 3.90 (m, 2H), 3.70 (m, 1H), 3.60 (m, 1H),3.41 (m, 1H), 2.99 (s, 3H), 2.54 (m, 1H), 2.28 (m, 1H); MS (ES+): m/z470 (M+1).

The compound (0.1 g, 0.2 mmol) as obtained above was suspended inmethanol (2 mL) and treated with ethereal HCl and the organic solventevaporated to yield the compound,(+)-trans-2-(2-chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-chromen-4-onehydrochloride.

Yield: 0.1 g (92.8%);

1H NMR (CDCl₃, 300 MHz): δ 8.02 (d, 2H), 7.83 (d, 1H), 6.64 (s, 1H),6.41 (s, 1H), 4.23 (m, 1H), 3.73 (m, 2H), 3.68 (m, 1H), 3.51 (m, 1H),3.39 (m, 1H), 2.99 (s, 3H), 2.54 (m, 1H), 2.31 (m, 1H).

In Vitro Studies

Materials and Methods: Two cervical cancer cell lines SiHa and HeLa(ATCC), positive for HPV genome were used for evaluation of Compound Aand Compound B. The cell lines were transfected with a plasmid vectorexpressing p53 protein fused with a fluorescent protein EGFP usinglipofectamine (Invitrogen, CA, USA). After 48 h of transfection thecells were maintained for 30-45 days in DMEM (Sigma Aldrich, catalogueno. D5546) supplemented with 10% FBS and 800 μg/mL Geneticin (Gibco BRLLife Technologies, Inc) for 30-45 days. The stably expressing cells wereexpanded and evaluated for the presence of fusion construct underfluorescent microscopy. After 24 h of transfection, the cells weretreated separately with Compound A and Compound B (henceforth referredto as treated cells) for 24 h. Cells were harvested and were used foranalysis of status of E6, E7, p53 by western blot and by RT-PCR. Cellsnot treated with the Compound A and Compound B (henceforth referred toas untreated cells) were used as controls. HPV negative C33A cell wasused as negative control.

The terms fresh medium and culture medium refer to DMEM (catalogue no.D5546).

Example 2 Analysis of Status of E6, E7, p53 by Western Blot

To prepare whole cell extracts for Western blot, the treated cells werelysed in lysis buffer (50 mM Tris-HCl (pH 7.4), 1% NP-40, 40 mM NaF, 10mM NaCl, 10 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride, and 10 mMdithiothreitol and 1 μg/ml each of leupeptin and aprotinin). The celllysates (50 μg) were resolved by SDS-PAGE and the separated proteins(E6, E7 and p53) were transferred to polyvinylidene difluoride membrane(PVDF) by wet transfer method using Bio-Rad electro-transfer apparatus.After blocking with 10% non-fat milk in Tris buffered saline containing0.2% Tween-20, the membrane was incubated with the primary antibody,followed by HRP-conjugated secondary antibody. Proteins were visualizedby 3,3′-Di amino benzidine (DAB) method. Similar procedure was followedfor untreated cells.

The following antibodies were used for western blot with the indicateddilutions.

Primary Antibodies

Sr. Source No. Antibody Organism Company Dilution 1 p53 Antibody RabbitCell Signaling (9282) 1:500 2 HPV16 E7(NM2) Mouse SantaCruz (sc-65711)1:100 3 HPV E76 (N-17) Goat SantaCruz (sc-1584) 1:100 4 Beta actin MouseSigma (A5316) 1:1000

Secondary Antibodies

Sr. No. Antibody Company Dilution 1 Rabbit anti goat HRP MolecularProbes (R21459) 1:2000 2 Anti mouse HRP Sigma (A9044) 1:2000 and 1:40003 Anti rabbit HRP Sigma (A0545) 1:2000

Results: It was observed that cells treated with Compound A and CompoundB showed increased expression of p53 protein in a dose dependant mannerin both SiHa and HeLa cells. However no expression was observed inuntreated cells. The expression of E6 was down regulated in SiHa cellsby both the compounds at the concentration of 50 μg/mL. Similarly thecomplete down regulation of E7 was seen in cells treated with 25 μg/mLof Compound A and also complete down regulation of E7 was seen in cellstreated with 25 μg/mL of Compound B compared to control (FIG. 1A andFIG. 1B).

Example 3 Analysis of Status of E6, E7, p53 by RT-PCR

The expression status for gene E6 and E7 at the transcription level wasdetected by Reverse Transcriptase PCR (RT-PCR). The RNA was isolatedfrom the treated and untreated cells using Qiagen AllPrep DNA/RNA MiniKit (Cat. No. 80204). The RNA integrity was checked by visualizationunder UV after agarose gel electrophoresis. The quantity and purity ofRNA was analysed by Nano Drop (Thermo Scientific). The 500 ng of RNA wasconverted into cDNA using 0.1 μg/μl of Oligo dT (Promega), MMLV-RT,dNTPs, RNA guard and RT buffer (Promega) and incubated at 37° C. for 1h. The reaction was terminated by heating the reaction mixture at 90° C.for 3 minutes and used for PCR amplification of E6 and E7 genes. The PCRwas conducted as follows:

5x Buffer 3 μl DNA taq polymerase 0.3 μl Antisense primer (10 pmol) 1 μlSterile water 5.45 μl 10 mM dNTPs 0.8 μl Sense primer (10 pmol) 1 μlMgCl₂ (50 mM) 0.45 μl cDNA 3 μlThe sequence of the primers are shown below

Gene Primer Sequence Size β-Actin sense 5′-AGACTTCGAGCAGGAGATG-3′(SEQ ID NO: 1) 256 bp Antisense 5′-CTTGATCTTCATGGTGCTAGG-3′(SEQ ID NO: 2) 16 E6 sense 5′-TGAGGTATATGACTTTGCTTTTC-3′ (SEQ ID NO: 3)297 bp Antisense 5′-CAAGACATACATCGACCGGTCC-3′ (SEQ ID NO: 4) 16 E7 sense5′-AAATGACAGCTCAGAGGAGGAG-3′ (SEQ ID NO: 5) 209 bp Antisense5′GTTTCTGAGAACAGATGGGGCAC-3′(SEQ ID NO: 6)

Results: RT-PCR quantification revealed significant inhibition oftranscripts for both E6 and E7 in both SiHa and HeLa cells treated with50 μg/mL of either Compound A or Compound B. The β-actin was used as ahouse keeping gene to normalize the data in RT-PCR assay. Thespecificity of gene specific PCR was determined using C33A cells whichare negative for E6 and E7 genes (FIG. 2).

Example 4 p53 Nuclear Translocation Assay

Materials and methods: Two cervical cancer cell lines SiHa and HeLa(ATCC), positive for HPV genome were used. The cell lines weretransfected with the expression vector containing p53-EGFP usinglipofectamine (Invitrogen, CA, USA). After 48 h of transfection thecells were maintained for 30-45 days in DMEM (Sigma Aldrich, catalogueno. D5546) supplemented with 10% FBS and 800 μg/mL Gen eticin (Gibco BRLLife Technologies, Inc) for 30-45 days. The stably expressing cells wereused for the p53 Nuclear Translocation Assay.

Biological Studies: The stably expressing cells were seeded on 96 wellimaging plates (Becton Dickinson, USA) and allowed to grow for 48 h in ahumidified CO₂ incubator at 37° C. The cell nucleus were stained withvital fluorescent nuclear dye Hoechst 22334 (0.5 μg/mL) for 5 minutesfollowed by fresh medium replacement. A stock solution of 50 mg/mL wasprepared in DMSO and further dilutions were made to obtain theconcentration of 2-50 μg/mL were made in the culture medium containing5% FBS. Cells were treated separately with different concentrations(2-50 μg/mL) of Compound A and Compound B. The imaging was carried outat every 12 h using pathway Bio imager (BD, USA) using 20× objective.The cells were segmented based on the nuclear channel and number ofcells positive for nuclear—EGFP were calculated to score activity.Alternatively the cells were also imaged with Nikon Ti invertedfluorescent microscope equipped with CCD camera Retiga Exi and NISelement software. The bright field images were also collected.

Results: Microscopic imaging for nuclear translocation of EGFP linkedp53 protein demonstrated that addition of Compound A and Compound Bresulted in increased nuclear translocation of EGFP-p53 protein in bothHeLa and SiHa cells. Further it was also noticed that nucleartranslocation was initiated at 12 h post treatment and increased up to24 h at 25 and 50 μg/mL concentration of Compound A as well as CompoundB. The representative graph showing the percentage p53 EGFP nuclearpositive cell for each drug for the different concentration of drug isshown in (FIG. 3A and FIG. 3B).

Example 5 Senescence Assay

This assay was performed to detect senescence associated β-galactosidasestaining to check the senescence status in the treated cells.

Materials and methods: Two cervical cancer cell lines SiHa and HeLa(ATCC), positive for HPV genome were used. The cell lines weretransfected with the expression vector containing p53-EGFP usinglipofectamine (Invitrogen, CA, USA). After 48 h of transfection thecells were maintained for 30-45 days in DMEM (Sigma Aldrich, catalogueno. D5546) supplemented with 10% FBS and 800 μg/mL Geneticin (Gibco BRLLife Technologies, Inc) for 30-45 days. The stably expressing cells wereused for the Senescence assay.

Biological Studies: The stably expressing cells were treated withCompound A and Compound B separately and fixed for 5 minutes in 3%formaldehyde, washed, and incubated at 37° C. with5-bromo-4-chloro-3-indolyl β-D-galactopyranoside solution (1 mg/ml),(prepared by dissolving in a solution containing 40 mM citric acid (pH6.5), 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mMNaCl, and 2 mM MgCl₂). After overnight incubation at 37° C., tissues orcells were visualized by microscopy. The intense blue staining in thecytosol indicated the induction of senescent like features in the cells.

Example 6 In Vivo Assay

All experiments were carried out in accordance with the guidelines ofCommittee for the Purpose of Control and Supervision of Experiments onAnimals (CPCSEA) and with the approval of Institutional Animal EthicsCommittee (IAEC) in Piramal Healthcare Limited, Goregoan, Mumbai, India.

In vivo efficacy of the compounds of the present invention for thetreatment of HPV associated cancer was studied by using SiHa (HPV 16positive cervical cancer) cell line.

SiHa Human Cervical Cancer Xenograft Mouse Model

Animals used: Nude Nu/J, Homozygous for FOXn1(Nu) male mice, 4 to 6weeks old, weighing 22 to 25 g (Harlan Laboratories, US). Animals werehoused in animal isolator under specified pathogen-free conditionsmaintained at 22 to 25° C. and 55 to 75% humidity, with a 12-hourlight/12-hour dark cycle. The mice were acclimatized for a period ofseven days before experimentation. Animals were handled in a laminarflow hood. All food and water was autoclaved.

Generation of Subcutaneous Xenograft Tumors in Nude Mice

Step 1: Preparation of a Single-Cell Suspension for Injection into NudeMice

The adherent SiHa cells (SiHa cell line-ATCC—HBT 35) were harvestedusing Trypsin-EDTA solution and suspension was centrifuged at 800 rpmfor 5 minutes. The supernatant was removed and the cell pellet wasre-suspended in 1 mL serum-free MEM medium. The cell count was obtainedusing 1:100 dilution. The cells were diluted to obtain 5×10⁶ cells per0.2 mL of suspension.

Step 2: Generation of Subcutaneous Xenograft Tumors in Nude Mice

On the day of tumor cell injection, cell suspension of step 1 was storedon ice in the laminar airflow hood. Each nude mouse was injected with0.2 mL of the cell suspension subcutaneously on the right flank. Theanimals were observed after 5 days for tumor growth by palpation aroundthe injection site.

Conditions for Storage of the Compounds and Dose Preparation

Compound A: 3.5 mg/mL; vehicle: dextrose (5%) prepared in water.

Topotecan Hydrochloride (used as standard): 0.2 mg/mL; vehicle: methylcellulose (0.25%).

Compound B: 20 mg/mL; vehicle: methyl cellulose (0.25%).

Cisplatin (used as standard): 0.6 mg/mL; vehicle: methyl cellulose(0.25%).

All the compounds including the standard were stored at 4° C. to 8° C.

Dosing

Nude mice were housed in a group of 8 per cage (filter-top cages) withautoclaved husk bedding and free access to food and water was provided.Animals were handled as per the standard guidelines. Treatment wasinitiated when tumor size volume was about 100 mm³. The tumor-bearingmice were randomized (n=8) in the following groups of treatment:

-   i) Group 1: Control group: Tumor-bearing mice administered with    vehicle.-   ii) Group 2: Tumor-bearing mice administered once daily with 35    mg/kg of compound A intraperitoneally.-   iii) Group 3: Tumor-bearing mice were administered with a single    dose of 2 mg/kg of Topotecan (standard) intraperitoneally.-   iv) Group 4: Tumor-bearing mice were administered once daily with    200 mg/kg of Compound B p.o.-   v) Group 5: Tumor-bearing mice were administered with a single dose    of 6 mg/kg of Cisplatin (standard) intraperitoneally.

Treatment

Nude mice of Group 1, Group 2 and Group 4 were treated on days 1-6 and12-17. Group 3 and Group 5 mice were treated on day 1. On the 23^(rd)day animals from all groups were scarified and samples were harvestedfor further analysis.

The volume administered to all the above Groups was 10 mL/kg.

Observations and Measurement

Following parameters were observed during treatment:

1. Gross animal health was observed everyday2. Body weight was observed everyday3. Tumor was measured 2-3 days apart, using vernier caliper.

Tumor volume in mm³ was calculated using the formula for a prolateellipsoid:

Tumor volume (mm³)=Length (mm)×[Breadth (mm)²]×0.5

-   -   assuming specific gravity of tumor as 1 and π as 3

Treated to control ratio (ΔT/ΔC %) on a given day was calculated usingthe formula:

${\Delta \; T\text{/}\Delta \; C\% \mspace{14mu} {on}\mspace{14mu} {Day}\mspace{14mu} X} = {\frac{\begin{matrix}{{{Tumor}\mspace{14mu} {size}\mspace{14mu} {Compound}_{{Day}\mspace{14mu} X}} -} \\{{Turmor}\mspace{14mu} {size}\mspace{14mu} {Compound}_{{Day}\mspace{11mu} 0}}\end{matrix}}{\begin{matrix}{{{Tumor}\mspace{14mu} {size}\mspace{14mu} {control}_{{Day}\mspace{11mu} X}} -} \\{{Tumor}\mspace{14mu} {size}\mspace{14mu} {control}_{{Day}\mspace{11mu} 0}}\end{matrix}} \times 100}$

Growth inhibition (GI) was calculated using the formula

GI on Day X=100−ΔT/ΔC% on Day X

Tumor growth inhibition results are given in FIG. 4 and FIG. 5.

Conclusion: Compound A and Compound B showed significant tumor growthinhibition in HPV mediated cervical cancer xenograft model.

1. A method for the treatment of human papillomavirus (HPV) associatedcancer comprising administering to the subject in need thereof atherapeutically effective amount of a compound of Formula (I), or apharmaceutically acceptable salt, a solvate, stereoisomer or adiastereoisomer thereof;

wherein Ar is a phenyl group, which is unsubstituted or substituted by1, 2, or 3 identical or different substituents selected from: halogen;nitro, cyano, C₁-C₄-alkyl, trifluoromethyl, hydroxyl and C₁-C₄-alkoxy.2. The method according to claim 1 wherein, in the compound of Formula(I) Ar is a phenyl group substituted by 1, 2 or 3 identical or differentsubstituents selected from chlorine, bromine, fluorine, iodine,C₁-C₄-alkyl and trifluoromethyl.
 3. The method according to claim 1wherein, in the compound of Formula (I) Ar is a phenyl group substitutedby chlorine.
 4. The method according to claim 1 wherein, in the compoundof Formula (I) Ar is a phenyl group substituted by chlorine andtrifluoromethyl.
 5. The method according to claim 1, wherein thecompound of Formula (I) is a (+)-trans isomer represented by Formula(IA), or a pharmaceutically acceptable salt or a solvate thereof;

wherein Ar is a phenyl group, which is unsubstituted or substituted by1, 2, or 3 identical or different substituents selected from halogen,nitro, cyano, C₁-C₄-alkyl, trifluoromethyl, hydroxyl or C₁-C₄-alkoxy. 6.The method according to claim 5, wherein the compound of Formula (IA) is(+)-trans-2-(2-Chlorophenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-3-yl)-chromen-4-onehydrochloride (compound A).
 7. The method according to claim 5, whereinthe compound of Formula (IA) is(+)-trans-2-(2-Chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-3-yl)-chromen-4-onehydrochloride (compound B).
 8. The method according to claim 1, whereinthe HPV associated cancer is selected from anal cancer, vulvar cancer,vaginal cancer, penile cancer, cervical cancer, oropharyngeal cancer,cancer of the oral cavity, lung cancer, non-melanoma skin cancer andcancer of the conjunctiva.
 9. The method according to claim 8, whereinthe HPV associated cancer is cervical cancer.
 10. A method for thetreatment of human papillomavirus (HPV) associated cancer comprisingadministering to the subject in need thereof a therapeutically effectiveamount of a pharmaceutical composition comprising a therapeuticallyeffective amount of the compound of Formula (I) as defined in claim 1,or a pharmaceutically acceptable salt, a solvate, a stereoisomer or adiastereoisomer thereof and a pharmaceutically acceptable carrier. 11.The method according to claim 10, wherein the pharmaceutical compositioncomprises at least one further pharmaceutically active compound, whereinthe pharmaceutically active compound has anticancer activity.